NPA Binding Sites Research Articles

5-Oxygenated N-Alkyl-and N,N-Dialkyl-2-amino-1-methyltetralins. Effects of Structure and Stereochemistry on Dopamine-D2-receptor Affinity

The ability of a series of stereochemically well-defined 5-oxygenated 2-aminotetralins, consisting of dopamine-receptor agonists and antagonists, to displace [3H]spiperone and [3H]N-propylnorapomorphine (NPA) from calf-caudate dopamine receptor sites has been evaluated in-vitro. In addition, the partition coefficients of the compounds were determined to measure their lipophilicity. The data were compared with previously obtained in-vivo biochemical data (dopa accumulation in reserpine pretreated or non-pretreated rats). Compounds with 2S-configuration and a C5-hydroxy substituent have the highest affinity for NPA-binding sites and such derivatives also have the highest potency in-vivo. The 2R-derivatives are less efficacious and their affinity for NPA- and spiperone binding sites is influenced by their lipophilicity. On the basis of these results, a model is proposed in which the antagonists form two, and the agonists form three, strong intermolecular bonds with the D2-receptor. According to this model, the agonists, but not the antagonists, are able to donate a hydrogen bond from the phenolic hydroxyl to the receptor.

Regulation of dopamine D2 receptor affinity by cholecystokinin octapeptide in fibroblast cells cotransfected with human CCKB and D2L receptor cDNAs.

Alteration in dopamine (DA) and/or cholecystokinin (CCK) transmission in the CNS may be of relevance for schizophrenia. Previous findings in striatal membranes give indications of a modulation of DA D2 receptor affinity by CCKB receptor activation. In the present study receptor binding studies were performed in a mouse fibroblast cell line (L-hD2l/CCK), expressing both human D2 receptors (long form, D2L) and human CCKB receptors, and binding sites for [3H]CCK-8S (sulfated CCK octapeptide), the D2 agonist [3H]NPA and the D2 antagonist [3H]raclopride were found and characterized in saturation and competition experiments. 1 nM of CCK-8 caused a significant 38% increase in the KD value of the D2 agonist [3H]NPA binding sites in the L-hD2l/CCK cell membranes. This change was blocked by the CCKB receptor antagonist PD 134308 (50 nM). Furthermore, 1 nM of CCK-8 increased the KD value of the D2 antagonist [3H]raclopride binding sites by 34% (P < 0.05) in the L-hD2l/CCK cell membranes. Control cells (L-hD2l cells) expressing D2L receptors showed no specific [3H]CCK-8S binding sites and no modulation by CCK-8 of the D2L receptors. These findings indicate a modulation of the D2L receptor affinity by activation of the CCKB receptor also when they are coexpressed in a fibroblast cell line. One possible explanation of these data may include a receptor-receptor interaction between the CCKB and D2L receptors.

A single (−)-nicotine injection causes change with a time delay in the affinity of striatal D 2 receptors for antagonist, but not for agonist, nor in the D 2 receptor mRNA levels in the rat substantia nigra

The in vitro and in vivo effects of (−)-nicotine on dopamine D 2 receptors in the rat neostriatum have been studied using biochemical binding, in situ hybridization and immunocytochemistry. A single i.p. injection (1 mg/kg) of (−)-nicotine resulted in a reduction of the K D value of the D 2 antagonist [ 3H]raclopride binding sites in rat neostriatal membrane preparations at 12 h without any significant change in the B max value. This action of (−)-nicotine was counteracted by pretreatment 15 min earlier with the nicotine antagonist mecamylamine (1 mg/kg, i.p.). However, the K D and the B max values of the D 2 agonist [ 3H]NPA binding sites in the rat neostriatal membrane preparations were not significantly affected 0.5–48 h after a single i.p. injection with 1 mg/kg of (−)-nicotine. No significant change in neostriatal D 2 receptor mRNA levels was observed at any time interval after the (−)-nicotine injection. No significant change was observed in tyrosine hydroxylase (TH) immunoreactivity in either the substantia nigra or the neostriatum, nor in nigral TH mRNA levels during the time interval studied (4–24 h posttreatment). Furthermore, addition of low (10 nM) or high (1 μM) concentrations of (−)-nicotine in vitro to rat neostriatal membranes did not alter the characteristics of [ 3H]raclopride or [ 3H]NPA binding. These results indicate that a single (−)-nicotine injection can produce a selective and delayed increase in the affinity of D 2 receptors for the antagonist, but not for the agonist without modifying the levels of D 2 receptor mRNA, probably via the activation of central nicotinic receptors.

Targeting of auxin carriers to the plasma membrane: effects of monensin on transmembrane auxin transport in Cucurbita pepo L. tissue

Treatment of etiolated zucchini (Cucurbita pepo L.) hypocotyl tissue with sub-micromolar concentrations of the cationophore monensin rapidly (<20 min) inhibited the transport catalytic activity of the specific auxin-anion efflux carrier and reduced the inhibition of this carrier by the phytotropin N-1-naphthylphthalamic acid (NPA). Monensin inhibited the basipetal polar transport of indol-3yl-acetic acid (IAA) in long (30 mm) zucchini segments. At concentrations lower than 10−5 mol·dm−3 monensin did not affect uptake of the ΔpH probe [2-14C]5,5-dimethyloxazolidine-2,4-dione (DMO) or that of the membrane-potential probe tetra[14C-phenyl]phosphonium bromide (TPP+), did not affect the response of IAA net uptake to external Ca2+ concentration and did not alter the metabolism of IAA. It was concluded that low concentrations of monensin inhibit transport through the Golgi apparatus of auxin efflux carrier protein and that the efflux carriers turn over very rapidly in the plasma membrane. Monensin pretreatment did not affect the saturable binding of [3H]NPA to microsomal membranes, indicating that the auxin-efflux catalytic sites and the NPA-binding sites are located on separate proteins. At higher concentrations (≤10−5 mol·dm−3) monensin inhibited both mediated uptake and mediated efflux components of IAA transport. This effect was at least in part attributable to perturbation by monensin of the driving forces for mediated uptake since high concentrations of monensin also reduced the uptake of DMO and TPP+.

Neurotensin Decreases the Affinity of Dopamine D2 Agonist Binding by a G Protein‐Independent Mechanism

To examine whether GTP-binding proteins (G proteins) mediate the ability of neurotensin to lower the affinity of dopamine D2 agonist binding, the modulation by neurotensin in vitro of N-[3H]propylnorapomorphine [( 3H]-NPA) binding was investigated following pretreatment with pertussis toxin and N-ethylmaleimide in rat neostriatal membranes. Preincubation with N-ethylmaleimide (100 microM) markedly inhibited pertussis toxin-induced back-ADP ribosylation of three proteins with apparent molecular masses of 41, 40, and 39 kDa, respectively. This inhibition was prevented by adding dithiothreitol (250 microM) during the preincubation. N-Ethylmaleimide increased the KD (180 +/- 30%) and decreased the Bmax (-31 +/- 9%) of [3H]NPA binding sites but did not affect the binding properties of the selective D2 antagonist [3H]raclopride. N-Ethylmaleimide pretreatment did not affect the neurotensin (3 nM)-induced increase in the KD of [3H]NPA binding sites. Pertussin toxin treatment in vivo and in vitro was similarly ineffective. In conclusion, the present study indicates that neurotensin modulation of D2 agonist binding in neostriatal membranes is not mediated by G proteins.

Neurotensin reduces the affinity of dopamine D 2 receptors in membranes from post mortem human caudate-putamen

The effects of neurotensin in vitro were investigated on the binding characteristics of N-[ 3 H]propylnorapomorphine ([ 3H]NPA) binding sites in crude membrane preparations from post mortem human caudate-putamen, obtained within 10 h after death from 3 patients without reported neurological or psychiatric diseases. Neurotensin (0.1–30 nM) was found to increase the K d value of [ 3H]NPA binding sites with a maximal increase of about 50% (420±45 pM) at 3 nM of neurotensin, without significantly affecting the B max value (123±20 pmol/g protein). These results indicate the existence of an intramembrane regulation by neurotensin receptors of D 2 receptors in the human caudate-putamen.

Changes in pituitary-adrenal activity affect the binding properties of striatal dopamine D-2 receptors but not their modulation by neurotensin and cholecystokinin-8

The effects of adrenalectomy were investigated on the binding properties of [ (3)H] N- propylnorapomorphine ([(3)H]NPA) binding sites in membrane preparations from the rat neostriatum. Adrenalectomy (1 week) was found to increase the K(D) value of [(3)H]NPA binding sites by 30 +/- 3%, without affecting the B(max) values. These effects were fully counteracted by replacement treatment with corticosterone (5 mg/kg, twice daily, 1 week, last injection given 2 h before killing). The ability of neurotensin and cholecystokinin-8 (sulphated) in vitro to increase the K(D) value of [(3)H]NPA binding sites was maintained also following adrenalectomy. The average increases were 16+/-7 and 21+/-6%, respectively. When added together the two peptides failed to increase the K(D) value any further. The B(max) values of [(3)H]NPA binding sites were not affected by either adrenalectomy or by the peptides. These results indicate a glucocorticoid regulation of the binding characteristics of dopamine D-2 receptors in the rat striatum. The mechanism mediating the effects of adrenalectomy may involve an altered expression or regulation of the D-2 receptor or of its connected G-proteins. Probably, it is different from the mechanism whereby the neuropeptides modulate the D-2 receptor, since the neuropeptide-induced modulation was also maintained following the changes induced by adrenalectomy.

Bisulfite interacts with binding sites of the auxin-transport inhibitor N-1-naphthylphthalamic acid

The affinity of the auxin-transport inhibitor N-1-naphthylphthalamic acid (NPA) for membrane particles as well as for solubilized binding sites from Cucurbita pepo L. hypocotyls was reduced by low concentrations of bisulfite (half-maximal inhibition at 2·10(-3)-3·10(-3) M). Two membrane fractions obtained by sedimentation aided with polyethylene glycol showed differential sensitivity to bisulfite. Other oxidizing or reducing substances tested at 1 mM had no effect, except for N-ethylmaleimide (80% inhibition) and iodine (complete inhibition), both of which reduced the number of binding sites but not their affinity. Addition of bisulfite to either the isoalloxane ring of flavoproteins or to pyridoxal phosphate or quinones is proposed as a possible mechanism of action. Sulfur dioxide, at concentrations measured in polluted air, can lead to bisulfite concentrations in plant tissue sufficient to interfere with NPA-binding sites and hence with auxin transport.

In situ molecular size of agonist dopamine D-2 binding sites in rat striatum.

Specific binding of [3H]N-propylnorapomorphine [( 3H]NPA) to 3,4-dihydroxyphenylethylamine (dopamine) D-2 receptors was investigated in rat striatum in vitro. For various dopamine receptor substances, the rank order of potency to inhibit [3H]NPA binding was spiroperidol greater than or equal to NPA greater than LY 171555 greater than SCH 23390 greater than SKF 38393. A single high-affinity binding site was found in membranes prepared in either Tris-citrate buffer or imidazole buffer; the affinity constants were 0.11 and 0.76 nM, respectively. The number of receptors (33 pmol/g wet weight) was independent of whether the membranes were prepared in Tris-citrate buffer or imidazole buffer and was similar to the number of receptors estimated by [3H]spiroperidol binding to dopamine receptors. Irradiation inactivation of frozen whole rat striata showed a monoexponential loss of [3H]NPA binding sites without a change in the binding affinity. The target size of the [3H]NPA binding site was 81,000 daltons, which shows that the functional molecular entity to bind the dopamine D-2 agonist was smaller than the molecular entity to bind the dopamine D-2 antagonist [3H]spiroperidol (target size, 137,000 daltons).

Autoradiographic localization and quantification of dopamine D 2 receptors in normal human brain with [ 3H]N-n-propylnorapomorphine

The precise distribution of dopamine receptors has been studied autoradiographically in the normal human brain using [ 3H]N-n-propylnorapomorphine ([ 3H]NPA) as a ligand. Preliminary experiments aimed at optimizing the binding assay conditions revealed that preincubation washing of caudate nucleus sections was a prerequisite to obtain a good ratio of specific to non-specific binding. The binding of [ 3H]NPA to caudate-putamen sections was saturable, stereospecific, reversible, of high affinity (K d = 0.27–0.35 nM) and occurred at a single population of sites. Competition experiments with various drugs indicated that in the caudate-putamen the specific [ 3H]NPA binding sites possess the pharmacological features of the dopamine D 2 receptor. The highest levels of [ 3H]NPA binding sites were found in the caudate nucleus, putamen, globus pallidus and nucleus accumbens. There were also intermediate to low concentrations of the 3H-ligand in the hippocampus, the insular and cingular cortices and in the occipito-temporal gyrus, while almost undetectable levels of binding were found in the anteior frontal cortex. Thorough examination of the subregional distribution of [ 3H]NPA binding sites in the caudate-putamen-pallidum complex revealed heterogenous patterns of radioactivity. In these brain regions, the distribution of autoradiographic grains was punctate and islands of high and low densities were observed. Moreover, in the caudate nucleus, there was a subtle high lateral to low medial gradient in the topography of the [ 3H]NPA binding sites and a more pronounced gradient along the rostrocaudal axis; the highest levels of binding being located at the midbody of the nucleus. No gradients of [ 3H]NPA binding were observed in the putamen. The present data indicate that [ 3H]NPA is a suitable ligand for accurate autoradiographic labeling of dopamine D 2 receptors in human postmortem brain tissue and that dopamine receptors are heterogeneously distributed and topographically organized in patches and gradients in the basal ganglia regions.

L-glutamate reduces the affinity of [ 3H]N-propylnorapomorphine binding sites in striatal membranes

l-Glutamate but not methyl-D-aspartate (NMDA) or quisqualate (Quis) (10 −6 M) in vitro with or without preincubation increased significantly the K D value of the [ 3H]N-propylnorapomorphine ([ 3H]NPA) binding sites by 21 and 36% respectively in striatal membranes of rat without influencing the striatal [ 3H]spiperone binding sites. The number of striatal [ 3H]NPA binding sites was not changed by l-glutamate (10 −6 and 10 −5 M) in vitro. There may thus exist interactions between striatal glutamate receptors — not related to excitatory amino-acid receptors of the NMDA or the QUIS type - and high affinity striatal DA receptors.

In vivo binding of N-n-propylnorapomorphine in the rat brain: Regional localization, quantification in striatum and lack of correlation with dopamine metabolism

The accumulation and retention of radioactivity in rat brain were studied after intravenous injection of the dopamine (DA) agonist [ 3H]N-n-propylnorapomorphine ([ 3H]NPA). Dose-dependent saturable accumulation of label was found in the striatum, nucleus accumbens and olfactory tubercle. DA agonists (apomorphine, N,N-dipropyl-5,6-ADTN) and antagonists (haloperidol, cis-flupenthixol) prevented this accumulation. Enhanced accumulation of radioactivity in the striatum was found after 6-OHDA lesions and short- and long-term treatment with reserpine. These results are an indication of specific NPA binding to presumably postsynaptically situated DA receptors. One hour after administration of the drug, the effect of NPA on striatal DA metabolism was not correlated with receptor saturation. Maximal numbers of in vivo NPA binding sites (about 30 and 22 pmol · g −1) in striatal tissue were calculated from independent measurements at 15 and 60 min after NPA injection. Regional distribution of radioactivity after a tracer dose of [ 3H]NPA was assessed in 35 brain areas and parts of the spinal cord. In addition to the already mentioned DA-rich areas receptor-specific NPA binding was also found in several other brain parts.

Behavioural and biochemical consequences of persistent overstimulation of mesolimbic dopamine systems in the rat

Rats, separated into high and low activity responders on the basis of their response, in terms of hyperactivity, to peripherally administered (−) N- n-propylnorapomorphine [(−)NPA] were subject to chronic infusion of dopamine or its solvent bilaterally into the mesolimbic system (nucleus accumbens) for 13 days via Alzet osmotic minipumps. Both high and low activity responders exhibited enhanced spontaneous locomotor activity during the infusion of dopamine (but not solvent) whether measurements were made over a 180-min period in individual photocell cages or over 24 hr via Automex activity meters in a grouped situation. The ability of (−)NPA to stimulate locomotor activity was virtually abolished in both high and low activity responders from the second day of the infusion of dopamine to its termination. This reduction continued for up to 42 days following withdrawal of dopamine from the high activity animals. In contrast, the animals initially classified as low activity responders gave markedly enhanced activity when challenged with (−)NPA 2–3 weeks after withdrawal from dopamine. Hyperactivity, caused both by infused dopamine and by peripherally administered (−)NPA, was shown to be selectively antagonized by neuroleptic agents. Solvent infusion did not alter behavioural responding from control either during or after infusion. Radioligand binding assays using[ 3H]NPA showed three clear differences between the two selected groups of rats. First, there were significantly more [ 3H]NPA binding sites in the mesolimbic tissue of normal high activity responders than normal low activity animals; secondly, chronic dopamine infusion increased [ 3H]NPA binding sites for low activity animals at a time when their responses to (−)NPA were markedly enhanced; thirdly, chronic infusion of dopamine decreased the numbers of [ 3H]NPA binding sites in high activity responders at a time when their responses to (−)NPA were markedly reduced.